The high genetic variability of human immunodeficiency virus (HIV) has seriously impaired the development of efficacious antiviral therapy. This hypervariability is driven by the high infidelity of retroviral replication which results in frequent mutation and the rapid selection of new variants. Many mutations confer altered host range, tissue specificity, cytopathogenicity, immunogenicity and/or drug sensitivity to the progeny virus and thus significantly affect disease progression. The importance of virus variation is evident, however, the mechanisms of retroviral mutation are poorly understood. Previously, we showed that purified HIV reverse transcriptase (RT) is error-prone in vitro and thus likely plays a role in generating new virus variants. However, more recent studies in our laboratory revealed that the accuracy of RT in vitro and in vivo differs significantly at select loci and that purified RT copies HIV genomic sequences in vitro with very low efficiency. Together, these data suggest that fidelity assays utilizing purified RTs in vitro are overly simplistic and do not fully represent the molecular events occurring during retroviral replication and mutation in vivo. We propose to better characterize the biochemical basis of retroviral mutation by comparing mutagenesis in cellular and cell-free systems. For these studies we will utilize Moloney murine leukemia virus (MLV)-based vector systems that are highly tractable for both in vitro and in vivo replication and mutation assays. MLV will serve as a model retrovirus and will be contrasted to HIV in future parallel studies as appropriate HIV vector systems are developed. The specific aims are: 1) to compare retroviral mutation rates and RT fidelity in forward mutation assays that genetically score mutations at several hundred independent loci; 2) to determine the influence of viral and cellular factor on RT fidelity by studying replication accuracy in permeabilized virions and by direct comparisons with mutations arising in cultured virus and errors catalyzed by purified RT in vitro at identical genetic loci; and 3) to determine the strand-specificity of replication errors both in vivo and in vitro and thus determine the relative contributions of RNA- and DNA-templated RT errors as well as RNA polymerase II infidelity to retroviral variation. Together, these studies will characterize the rates and preferred types of mutations arising in a model retrovirus and will identify the roles of viral and cellular factors in retroviral mutagenesis. An understanding of the mechanisms of retroviral mutations is fundamental to an understanding of the evolution of retroviruses and their facility to escape immunosurveillance and antiviral drugs. Moreover, a characterization of the biochemical basis of retroviral mutation and the molecular factors involved in these frequent events may identify novel strategies for perturbing retroviral mutations rates and thereby delaying HIV drug resistance or accelerating mutational suicide of progeny virus.